Affiliations
Lankenau Institute for Medical Research,
Wynnewood, Pennsylvania, United States of America,
Immunome, Inc., Wynnewood, Pennsylvania,
United States of America,
Augmenta Biologicals, LLC, Wynnewood,
Pennsylvania, United States of America

Figures

Abstract

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the
neuromuscular junction. The ideal countermeasures for BoNT exposure are
monoclonal antibodies or BoNT antisera, which form BoNT-containing immune
complexes that are rapidly cleared from the general circulation. Clearance of
opsonized toxins may involve complement receptor-mediated immunoadherence to red
blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing
immunoadherence of BoNT-specific antibodies may increase their potency
in vivo. We designed a novel fusion protein (FP) to link
biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP
consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT
complexes bound specifically to the RBC surface in vitro. In a
mouse model of BoNT neutralization, the FP increased the potency of single and
double antibody combinations in BoNT neutralization. A combination of two
antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice.
Neutralization in vivo was dependent on biotinylation of both
antibodies and correlated with a reduction of plasma BoNT levels. In a
post-exposure model of intoxication, FP:mAb complexes gave complete protection
from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure.
In a pre-exposure prophylaxis model, mice were fully protected for 72 hours
following administration of the FP:mAb complex. These results demonstrate that
RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT
neutralization by antibodies in vivo.

Funding: This work was supported by a grant from the National Institutes of Health, NIAID
R01 AI065967 to S.K.D. and institutional support from the Lankenau Institute for
Medical Research. Augmenta, LLC, provided support for PMS and contracted Impact
Biologicals, Inc. for production of the FP. PMS, as a corresponding author,
participated in study design, data collection and analysis, decision to publish,
and preparation of the manuscript.

Competing interests: The authors have read the journal's policy and have the following conflicts:
SPA and RB are part-time employees of Immunome, Inc., a company that has
optioned rights to the antibodies described in this report. BHS is the president
of Impact Biologicals, Inc. PMS is the inventor of the FP and the President of
Augmenta, LLC. SKD is a founder and Chief Scientific Officer of Immunome, Inc.
and has an equity interest in the company. SKD does not receive research support
or consulting fees from Immunome, Inc., Augmenta Biologicals, LLC, or Impact
Biologicals, Inc. A provisional US Patent application has been filed on the
experiments described, "Toxin Clearance", Inventors SPA, SKD, PMS, and has been
assigned to LIMR and Augmenta, LLC. These relationships do not alter the
authors' adherence to all the PLoS ONE policies on sharing data and
materials.

Introduction

Botulinum neurotoxin is one of the most potent lethal substances known. It is
produced by organisms of the genus Clostridium and produces
peripheral neuromuscular and autonomic paralysis through inactivation of cholinergic
signaling at the neuromuscular synapse. Intoxication with BoNT proceeds by a series
of steps, in which BoNT first enters the body, transits across an epithelium,
travels through the bloodstream, and interacts with the surface of cholinergic
neurons [1],
[2],
[3]. Once
bound to the neuromuscular junction, BoNT is internalized via binding to secretory
vesicle proteins and transported into a vesicular compartment. The catalytic domain
of BoNT, the light chain (LC), acquires proteolytic activity as it is transported
across the vesicle membrane into the neuron cytosol [4], [5]. Through cleavage of tethering
proteins, the BoNT LC prevents the neuron from releasing acetylcholine in response
to neural stimulation.

Passive immune therapies for BoNT intoxication have been shown to be effective
clinically and in laboratory studies, with either antisera or oligoclonal
combinations of monoclonal antibodies [6], [7], [8]. Within the bloodstream,
BoNT-containing immune complexes that contain three or more antibodies are rapidly
sequestered in the spleen and liver [3], [8]. Such clearance is
sufficient to provide high level neutralization (>10,000 LD50 BoNT), even if the
antibodies do not have intrinsic neutralizing activity [9], [10]. Immune complexes formed
between BoNT and only one or two antibodies stably circulate in the bloodstream and
are therefore much less potent in BoNT neutralization (L.L.S., data not shown).

A general feature of the handling of immune complexes in vivo is
immunoadherence, i.e., attachment to red blood cells (RBC) [11]. The precise mechanism for BoNT
clearance by immune complexes has not been elucidated, but it may involve multiple,
redundant systems for antigen capture by Fcγ receptor-bearing
reticuloendothelial cells in the liver and spleen [8], [12], [13]. One aspect of this process
utilizes the complement system, in which C3b-opsonized immune complexes bind to
complement receptor type 1 (CR1) on RBCs in primates or to complement factor H in
rodents [14],
[15]. The
ability of a monoclonal antibody to utilize this pathway can be enhanced by linking
it to another antibody specific for CR1, to create a bispecific
“heteropolymer” [16], [17]. Heteropolymer:antigen complexes bound to RBCs can be
directly taken up by macrophages and are rapidly cleared from the circulation.

Methods that enhance the immunoadherence of antibodies to RBCs may be useful for BoNT
prophylaxis and treatment. Antibody immunoadherence may be enhanced using a novel
fusion protein (FP), created by Augmenta Biologicals (Wynnewood, PA). The FP is a
recombinant protein that links streptavidin [18] to an scFv derived from a
monoclonal antibody specific for GPA, the predominant protein on the RBC surface
[19]. The FP
was developed as a delivery system to adhere biotinylated molecules to the RBC
surface, which may enhance the immunogenicity of biotinylated vaccine antigens and
the clearance of biotinylated antibody-antigen complexes. We previously described a
panel of human monoclonal antibodies specific for BoNT serotypes A and B (BoNT/A,
BoNT/B) [20],
[21], [22]. In this study,
we examined the ability of the FP to augment the neutralizing capability of these
antibodies in vivo.

Results

Binding of FP:mAb:BoNT complexes to red blood cells in
vitro

The FP is a recombinant protein that joins an N-terminal scFv specific for GPA to
a C-terminal streptavidin moiety (Figure 1a). GPA is expressed exclusively on the RBC
membrane, at approximately 106 copies per cell, where its primary
role is to provide negatively charged sialic acid residues that limit RBC-RBC
aggregation [19]. The murine scFv sequence specific for GPA was
derived from the antibody TER-119 [23]. Streptavidin is a
tetrameric protein that binds biotin with high affinity [24]. The FP also contains a
C-terminal polyhistidine tag to facilitate purification following expression in
E. coli. Figure 1b shows a poly-acrylamide gel in which
urea-solubilized and refolded FP samples were analyzed. Two bands were seen in
the refolded FP sample, consistent with existence of tetrameric (164 kDa) and
monomeric (41 kDa) forms. We have observed that the monomeric FP is unable to
bind biotin (P.M.S., data not shown), consistent with the observations of others
[24].

(a) Schematic representation of the FP. The FP is comprised of an scFv,
specific for the RBC surface protein glycophorin A (GPA), fused to
streptavidin (StAv). The latter is capable of binding biotinylated mAbs
specific for BoNT. (b) SDS-PAGE of the FP performed without heating the
samples prior to loading. Lane 1: FP after expression in E.
coli and purification in 8M urea (monomer). Lane 2:
refolded FP following dialysis for removal of urea showing the tetramer
and residual monomer.

As depicted in Figure
1a, the FP was designed as a molecular bridge to link
biotinylated molecules, such as antigens and antibodies, to the RBC membrane. We
analyzed binding of the FP to the surface membrane of murine RBCs in
vitro using flow cytometry, labeling the FP with biotinylated
fluorescein. Figure
2a shows near complete labeling of the RBCs mediated by the FP
molecule. FP binding was specific for GPA, since its binding was completely
inhibited by the TER-119 IgG, but not by an isotype control antibody (rat
IgG2b). Next, we tested RBC binding of complexes containing FP, the
BoNT/A-specific MAb 6A, and BoNT/A 50 kDa C-terminal domain (HC50). The HC50 was
labeled with Alexa Fluor 488, and the biotinylated 6A MAb was detected with an
anti-human IgG-APC secondary antibody. In vitro incubation of
this complex with RBCs resulted in almost complete co-labeling of RBCs with APC
and Alexa-488 (Figure
2b, panels A and B). This binding was also
inhibited by TER-119, but not by the isotype control antibody (Figure 2b,
panels C and D).

(a) In-vitro RBC binding by the FP complex is specific
for glycophorin A. FP with or without biotinylated-fluorescein
(BIOT-FLUO) was incubated with excess competitor TER-119 antibody (TER)
or an isotype control (IgG) and analyzed by FACS. NL, no label. (b) RBC
binding of the FP:6A:BoNT complex. A) Unlabeled RBCs. B) FP,
biotinylated 6A and Alexa488-labeled HC50A were added to RBCs and
detected with an anti-human-APC antibody. C) Competitor TER-119
inhibited binding of the complex to RBCs. D) An IgG isotype control
antibody did not affect complex binding.

In vivo neutralizing ability of single antibodies bound to
toxin

We previously reported three human antibodies that are specific for BoNT [20], [21]. 4LCA binds
to the catalytic light chain domain of BoNT/A, and can neutralize 25 LD50 BoNT/A
in the standard mouse protection assay. The 6A and 13A mAbs bind to overlapping
epitopes on the BoNT/A heavy chain C-terminal 50 kDa domain (HC50). The 6A MAb
can neutralize 2.5 LD50 BoNT/A in vivo, while the 13A MAb is
essentially non-neutralizing. The 30B MAb binds serotype B BoNT (BoNT/B) with
high affinity, but it is not neutralizing. We biotinylated these mAbs and tested
them in vivo alone or in combination with excess FP. The most
effective complex contained 6 µg FP and 1.5 µg 4LCA, which was able
to completely neutralize up to 100 LD50 BoNT, 4-fold greater than could be
neutralized by 100 µg naked 4LCA MAb (Table 1). The FP also enhanced
the activity of the 6A MAb, allowing neutralization of 25 LD50 BoNT/A, a 10-fold
increase over the 2.5 LD50 neutralized by 100 µg naked 6A. Control
experiments with un-biotinylated 4LCA and 6A mAbs, administered with FP, showed
no enhancement in activity (data not shown). Incorporated into an FP complex,
the non-neutralizing 13A MAb protected mice up to a dose of 10 LD50. Similarly,
the non-neutralizing 30B MAb could neutralize BoNT/B in vivo
when bound to the FP, although the dose of complex was larger (12 µg FP, 3
µg 30B) and the amount of toxin was smaller (5 LD50). These observations
indicate that the FP can enhance the neutralizing activity of BoNT mAbs. In
addition, while FP quantitatively enhanced the potency of mAbs with intrinsic
neutralizing activity, it also converted qualitatively non-neutralizing mAbs to
neutralizing ones.

A recurrent finding of BoNT neutralization in vivo is that
oligoclonal antibody mixtures are more potent than single antibodies. We tested
a combination of the 4LCA and 6A mAbs, which together, in their unmodified
forms, can neutralize up to 1000 LD50 [22]. Using 0.75 µg each
MAb with 6 µg FP, we observed complete survival with up to 2000 LD50 BoNT,
whereas 50 µg each of naked 4LCA and 6A was not protective (Table 2).
Increasing the quantity of FP:Ab 4-fold (24 µg FP with 3 µg each
MAb), provided complete survival with BoNT doses of up to 5000 LD50. Mice that
received this dose and 10,000 LD50 BoNT/A survived one day following the
injection, indicating partial protection.

We next used the 2000 LD50 dose to test the importance of linking the MAb to the
RBC surface through the FP. Unbiotinylated 4LCA, at either 1.5 µg or 100
µg, did not contribute to neutralization by biotinylated 6A and the FP
(Table
2). When both mAbs were biotinylated, but given in combination
with streptavidin, rather than FP, no protection was seen. These results suggest
that efficacy in vivo requires the formation of a complex that
anchors both mAbs to the RBC surface by binding to one or more FP molecules. To
explore this further, we assessed the plasma BoNT levels in mice injected with 6
µg of detoxified BoNT/A and the 4LCA and 6A FP:mAb complexes (3 µg
each antibody, 24 µg FP) (Figure 3). Ninety minutes after injection, we
tested the levels of BoNT/A in undiluted plasma using ELISA. The presence of the
biotinylated mAbs alone reduced the BoNT levels slightly, but this effect was
significantly enhanced by the presence of the FP. Taken together, these results
demonstrate the collaborative effects of a pair of mAbs in combination with the
FP, and suggests that the neutralizing activity requires BoNT immunoadherence to
RBCs in vivo.

Figure 3. Plasma BoNT/A concentrations in mice injected with biotinylated
anti-BoNT antibodies with or without FP.

Groups of three mice each were injected i.v. with 6 µg of
inactivated BoNT/A, in the presence of the indicated combinations of
biotinylated mAbs 6A and 4LCA (3 µg each) and FP (24 µg).
After 90 minutes, blood was collected and plasma BoNT/A concentrations
were determined by ELISA.

Post-exposure and pre-exposure neutralization of BoNT by FP:mAb
complexes

We next assessed the FP:Ab mixture in post-exposure and pre-exposure models,
testing 6 µg FP with 0.75 µg each of the 4LCA and 6A antibodies. In
the post-exposure model, BoNT (10 LD50) was delivered by intraperitoneal (i.p.)
injection and FP:mAb complexes were subsequently administered at hourly
intervals by intravenous (i.v.) injection. Mice were monitored for survival for
5 days. Complete survival was provided by FP:Ab given up to 2 hours following
the BoNT injection, with partial survival at 3 and 4 hours post-BoNT
(87.5% and 62.5% survival, respectively; Figure 4). We also performed a
pre-exposure challenge with this dose of FP:mAb. Mice first received an i.v.
injection of the FP:mAb and were then challenged at daily intervals with 10 LD50
BoNT/A i.p. (Figure
5). FP:mAb complexes were able to provide complete survival
for mice receiving BoNT up to 72 hours after FP:mAb administration. Mice
receiving BoNT 96 hours after the FP:mAb were partially protected (40%
survival). Thus, the FP:mAb combination can provide protection against a lethal
dose of BoNT in both post-exposure and pre-exposure conditions.

Mice received i.v biotinylated mAbs 6A and 4LCA (0.75 µg of each)
and 6 µg FP. They were subsequently challenged with 10 LD50 BoNT
i.p. at the indicated times. Data are plotted as the percent of mice
surviving 5 days after the BoNT exposure. All mice that received the
FP:mAbs combination up to 72 hours prior to BoNT survived. Mice given
BoNT 96 hours after the FP:mAbs combination showed partial protection
(40% survival).

Discussion

The primary obstacles to the development of a comprehensive immune therapeutic for
BoNT exposure are the extreme potency of the toxin and the broad diversity of BoNT
serotypes. Experiments with polyclonal antibodies and oligoclonal antibody
combinations have shown that effective clearance of BoNT from the circulation is one
of the most important contributors to neutralization [1], [3], [8], [22], [25], [26]. The development of single or
double molecule therapeutics for BoNT will require enhancement of the ability of
mAbs to clear BoNT from the blood circulation.

In this study, we tested the ability of a novel FP to enhance the neutralizing
capacity of BoNT-specific mAbs at the level of the blood circulation. The FP is a
bifunctional molecule that allows the adherence of biotin-conjugated immune
complexes to the RBC surface. The FP was able to significantly increase the
neutralization potency of BoNT-specific mAbs. When combined with the FP, the
non-neutralizing 13A and 30B mAbs were able to fully neutralize lethal doses of
BoNT/A and BoNT/B, respectively. The FP increased the quantity of BoNT/A that could
be neutralized by the 6A mAb, the 4LCA mAb, and the 6A/4LCA mAb combination. These
levels were achieved with much lower quantities of mAbs than we had previously
required for protection with naked mAbs (1.5 µg vs. 50-100 µg). The NIH
and the FDA have not yet established a standard for protective efficacy for BoNT
passive immune therapies, but we can compare the potency of our FP combinations with
the approved BoNT anti-toxins. The human antiserum for use in infants, BabyBig
(MassBioLogics, Boston, MA), has a BoNT/A-specific potency of 3,000 LD50/mg [27]. Two of our
single FP:mAb combinations (with 6A or 4LCA) and our double MAb combination (with
both 6A and 4LCA) were equivalent or better in potency (3,333–266,666 LD50/mg,
Table 3). The
investigational heptavalent botulinum antitoxin (HBAT, Cangene Corporation,
Winnipeg, Manitoba, CA) has a potency of 7,500 IU/dose (1 IU equals 10,000 murine
LD50s) [28]. An
equivalent dose of the FP:mAb combination would amount to 281 mg, or 4 mg/kg in a 70
kg person. Thus, the potency of our FP:mAb combination is within the range required
for a human therapeutic.

Animal testing of a BoNT countermeasure needs to address protection of an
asymptomatic individual (who has absorbed a sub-lethal dose, but may still be
absorbing toxin) or an individual at risk for exposure (such as a first responder to
a contaminated area). We found that the FP combined with 6A and 4LCA is sufficient
to provide neutralization of 10 LD50 BoNT/A for 72 hours and partial protection at
96 hours. This result indicates the presence of physiologically relevant
concentrations of the FP:mAb combination in vivo during this
time.

We also tested the FP:mAb combination in a post-exposure model, in which the FP:Ab
was administered following a lethal dose of toxin. An intravenous injection of the
combination was able to provide complete protection from a lethal intraperitoneal
BoNT dose at 2 hours and partial protection at 4 hours. BoNT distribution
experiments have demonstrated that the window of opportunity of exposure of a lethal
dose of BoNT is determined by a first-order reaction that depends on the amount of
BoNT administered and the period of time that elapses before the countermeasure is
administered (L.L.S., manuscript in preparation). Thus, anti-toxins that are
sufficient to neutralize an entire circulating dose of BoNT should give the same
window of opportunity for post-exposure salvage, and our post-exposure results are
comparable to results obtained by others [10], [28], [29].

The proposed mechanism through which the FP augments antibody neutralization activity
involves binding of FP:mAb complexes to the RBC surface. Flow cytometry experiments
in vitro demonstrated that the FP:mAb complexes are able to
bind to erythrocytes and that the complexes serve as a molecular bridge to adhere
BoNT to the RBC. Experiments with un-biotinylated antibodies and the FP did not show
any enhancement of neutralization. Streptavidin alone did not improve neutralizing
activity of biotinylated BoNT antibodies, in comparison to the RBC-targeted FP.
Lastly, the levels of BoNT circulating in the plasma of mice that had received the
FP in combination with the 4LCA and 6A were lowered. These results together support
the model that the enhancement of neutralization in vivo required
biotin-dependent interaction of the cloned mAbs with the FP and adherence of the
FP-containing complexes to the surface of the RBCs (Figure 1a).

The observation that the intrinsic neutralization capacity of the naked mAbs
correlated directly with the potency level achieved when bound to the FP supports
the idea that the FP:mAb:BoNT complexes remain in circulation for a significant
period of time before they are definitively removed. In this model, relatively rapid
adherence of FP:mAb:BoNT to the RBC membrane would be followed by a slower phase, in
which either complex-bound toxin is removed from the RBC surface or the BoNT-bound
RBCs are removed from the circulation.

This is distinct from clearance of C3b-opsonized immune complexes, which are
definitively taken up by the liver and spleen in less than 15 minutes [8], [13]. While
circulating and adherent to RBCs, the FP:mAb complexes would be in competition with
the neuromuscular junction for BoNT. Intoxication may result from dissociation of
BoNT from the antibody complex, or of the FP:mAb:BoNT complex from the surface of
the RBC. The high potency of the FP:6A/4LCA complex may partly result from
stabilization of BoNT on the RBC surface through cooperative mAb avidity effects, as
maximal neutralization with the 4LCA and 6A antibodies was only observed when both
were biotinylated. Accelerated RBC destruction is not likely to be a factor in BoNT
clearance, as mice treated with FP do not exhibit a reduced hematocrit (data not
shown).

Our study has shown the value of immunoadherence as an effective mechanism for
improving the neutralizing ability of BoNT mAbs. The potency of the FP:mAb complexes
and their utility in the pre-exposure setting demonstrated that immunoadherent
immune complexes could be used to protect those at risk of BoNT exposure, in
addition to those already exposed. In practice, the FP could be held in a biodefense
stockpile as a non-specific immune adjuvant, to be combined with biotinylated MAb
specific for the toxin(s) to which people have been exposed. Alternatively, FP
sequences could be used to create hybrid MAb molecules that combine, in a single
polypeptide, RBC immunoadherent and anti-toxin activities in a single construct. An
important advantage of the FP is that it can be ligated quickly and irreversibly to
any molecule that has been biotinylated. This allows the creation of immunoadherent
complexes without having to synthesize novel fused polypeptides or add synthetic
linkers. Such experimental flexibility will facilitate the study of factors that
affect the potency, distribution and metabolism of different FP-containing complexes
in vivo in order to optimize their function as an accessory
immunotherapeutic agents.

Materials and Methods

Fusion protein construction and purification

DNA for the fusion protein scFv (provided by Dr. James Atkinson, Washington
Univ., St. Louis, MO) was fused in frame with coding sequences for core
streptavidin (provided by Dr. Stephan Dubel, Technical Univ. of Braunschweig,
Germany, based on Pahler, et al. [18]), followed by a
polyhistidine sequence and inserted into pET 21a(+).
BL21(DE3)pLysS cells (Invitrogen, Carlsbad, CA) were
transformed with the resultant plasmid and expression of the recombinant protein
was induced with IPTG. FP was purified from bacterial lysate using an
SP-sepharose and a His-Select Nickel Affinity gel (Sigma-Aldrich) and eluted
with TUB/100 buffer (60 mM Tris-HCl, 8 M urea, 100 mM imidazole, pH 8.0). FP was
dialyzed in a series of buffers containing 50 mM Tris-HCl and 0.4 M arginine,
slowly decreasing the urea concentration. The final preparation is in a buffer
of PBS-Arg, pH 7.4 (5 mM NaH2PO4, 70 mM NaCl, 0.4 M
arginine).

in vitro analysis of FP complexes binding to RBCs via flow
cytometry

Heparinized RBCs collected from female Swiss Webster mice (Taconic Farms, Hudson,
NY) were diluted 1∶2 in PBS:heparin (100 U/ml) and aliquoted at
106 per well and washed with 200 µl PBSA (PBS/1%
BSA). RBCs were incubated with or without 10-fold excess (4.4 mg) rat anti-mouse
TER-119 or rat IgG2b isotype control (eBiosciences, San Diego, CA) and incubated
at room temperature (RT) for 30 min. Cells were spun down at 2000 rpm in an
Allegra 6R centrifuge with a GH3.8 rotor (Beckman-Coulter, Brea, CA) 5 min and
incubated with 400 ng FP in 100 ml PBSA for 45 min. The mAb 6A, biotinylated or
un-biotinylated, was incubated with A1-Alexa488 for 1 hr at RT, and then added
to RBCs and incubated for 30 min. Cells were washed twice in PBSA and
F(ab')2 donkey anti-human IgG APC (Jackson ImmunoResearch, West Grove, PA)
added at 1∶10,000 and incubated at RT for 30 min. Cells were washed twice
in PBSA and resuspended in a final volume of 1 ml PBSA and analyzed on a BD
FACSCantoII (Becton Dickson, Franklin Lakes, NJ) using FlowJo 8.8.6. software
(Tree Star, Ashland, OR). Competition experiments were performed as above by
incubating 400 ng FP with biotin-fluorescein (Thermo Fisher Scientific) in
10-fold molar excess for 50 min, adding to RBCs pre-incubated with TER-119 as
described previously for 30 min, washing twice and analyzing by FACS.

A codon-optimized BoNT/A igene was constructed (GenScript USA, Piscataway, NJ).
The gene contained 4 point mutations, R363A and Y365F, which abolish the
catalytic activity of BoNT/A [30], and W1266L and Y1267S, which prevent binding of
BoNT/A at the neuromuscular junction [31]. The gene also encoded an
N-terminal polyhistidine tag followed by an enterokinase site. The gene was
inserted into the pTRCHisA vector, yielding the expression plasmid
pTRC-detoxBoNT/A, and expressed in E. coli BL21-codon plus(DE3)-RIL (Agilent
Technologies, Santa Clara, CA). Cells were grown in Terrific Broth (1.2%
peptone, 2.4% yeast extract, 0.94% K2HPO4 and 0.22% KH2PO4)
(Difco; Sparks, MD) at 37°C to ∼0.8 OD600, at which time IPTG was added,
the culture was cooled to 20°C in an ice bath, and the cells were incubated,
shaking, for 12 hours.

Purification of protein

Bacterial cells from 1 liter of culture were suspended in 200 ml of bacterial
protein extract reagent, B-PER (Pierce; Rockford, IL) at 4°C. Lysozyme
(Sigma; St. Louis, MO) at a final concentration of 0.1 mg/ml, DNASE (Sigma) at a
final concentration of 0.01 mg/ml, and protease inhibitor cocktail tablet
(Roche; Manheim, Germany) were added to the cell suspension and incubated on a
rotating shaker for 2 hr. Four hundred ml of 50 mM sodium phosphate containing
300 mM NaCl, pH 8.0, was added to the lysed cell suspension and allowed to stand
for 30 min. The suspension was centrifuged at 27,000 x g for 40 min to remove
precipitate.

The clear supernatant was loaded onto a 4 ml cation exchange column of CM
Sepharose fast flow (Amersham Bioscience; Piscataway, NJ) equilibrated with 50
mM sodium phosphate, pH 6.8. The column was washed with 50 volumes of 50 mM
sodium phosphate, pH 6.8. Bound protein was eluted from the column with a
stepwise increasing of NaCl concentration (10, 20, 40, 60, 100, 150, 200, 300
and 500 mM of NaCl with 50 mM sodium phosphate pH 6.8). The active fractions (at
∼200 mM NaCl) that correspond to about 150 kDa protein on a 10% SDS
polyacrylamide gel electrophoresis were pooled and dialyzed against PBS. The
purity of detoxified BoNT/A was confirmed on the SDS-PAGE and found to be more
than 98% homogeneous. The identity of the BoNT/A was confirmed by Western
blot analysis using rabbit polyclonal antibodies raised separately against the
catalytic domain (LC) and the heavy chain domain (HC50) of pure BoNT/A
toxin.

Digestion with enterokinase and generation of dichain detoxified
BoNT/A

C. botulinum produces BoNT/A as a di-chain active protein molecule (nicked).
Recombinant detoxified BoNT/A purified from E. coli was treated with a protease
enterokinase (EK) to make it a di-chain ‘nicked’ molecule. One
milligram of purified detoxified BoNT/A was incubated with five units of
enterokinase in a 1.5 ml of EK-Max buffer as described in EK-MaxTM kit
(Invitrogen) for O/N at 25°C. EK was then removed by EK-away TM resign
(Invitrogen). The digested protein sample was then diluted with 5 bed volumes of
50 mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole (pH 8.0). The solution
was centrifuged at 27,000 x g for 40 min to remove any precipitate.

The clear supernatant was passed through a 2 ml column of Ni-NTA superflow
(Qiagen) equilibrated with 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. The
N-terminal polyhistidine tag that was cleaved of from the detoxified BoNT/A
protein molecule was trapped by Ni-NTA resin and the remaining digested protein
molecule passed through the column. The pass-through sample was dialyzed against
PBS and concentrated using 30K ultracentricon (Millipore). The di-chain nicked
detoxified BoNT/A was compared with the native BoNT/A by reducing SDS-PAGE and
found to have two bands (a 50 kDA light chain and a 100 kDa heavy chain).

Blood plasma BoNT ELISA determination

For measurement of clearance of BoNT from the circulation by FP, the detoxified
BoNT/A was used. 6 µg of detoxified BoNT/A was incubated with 3 µg
biotinylated 6A, 3 µg biotinylated 4LCA, and 24 µg FP for 1 hr at RT
prior to tail vein injection in 25 g female Swiss Webster mice. Control mice
were given toxin only or no injection. Mice were anesthetized under isofluorane
90 mins after toxin administration and whole blood was collected by cardiac
puncture with a heparinized syringe. Blood was separated into microfuge tubes
and spun for 5 minutes at 3,000 RPM in a microcentrifuge for 5 minutes. The
plasma was aliquoted and stored at −20°C until use. To assay BoNT/A in
plasma, black 96-well flat bottom (Nunc Maxisorp) plates were coated at 4°C
overnight with 100 µl/well of 3B3, a human mAb that binds BoNT/A, at 2
µg/ml in PBS. Plates were washed with PBS/0.05% Tween-20
(Sigma-Aldrich, St. Louis) and then blocked for 1 h at 37°C with
PBS/0.05% Tween-20/5% non fat dry milk. Undiluted samples were
added at 100 µl/well, incubated for 2 hours at 37°C, washed, and
followed by addition of rabbit anti-HC50A serum at 1∶100 dilution. After
one hour, goat anti-rabbit-HRP was used at 100 µl/well (1∶10,000
dilution) and incubated for an additional hour. OPD was used as the colorimetric
substrate; optical density at 490 nm was measured.

Botulinum neurotoxins

BoNT/A1 and BoNT/B were obtained from Metabiologics (Madison, WI). LD50
eqivalents were 2.5 pg/LD50 for BoNT/A1 and 5 pg/LD50 for BoNT/B.

Ethics statement

All animal work was conducted according to all relevant guidelines in a protocol
approved by the Institutional Animal Care and Use Committee of the Lankenau
Institute for Medical Research, covered by protocol number A08-2692, Approval
Date: August 26, 2008, last amendment approval date, July 15, 2009, Animal
Welfare Assurance number A3550-01.

Acknowledgments

We are grateful to the laboratories of Lance Simpson at Thomas Jefferson University
and of George Prendergast and Scott Dessain and to the Lankenau Institute for
Medical Research.